1. Field of the Invention
The present invention generally pertains to the fields of molecular biology, protein crystallization, x-ray diffraction analysis, three-dimensional structural determination, rational drug design and molecular modeling of related proteins. The invention provides methods for determining the structure or function of one or more domains of a cation-dependent (and preferably calcium-dependent) polypeptide in the presence of one or more cations. The invention further provides methods for identifying a ligand having the ability to bind to one or more ligand-binding domains (LBDs) of a cation-dependent (and preferably calcium-dependent) polypeptide, and ligands identified by these methods. Finally, the invention provides methods of treating or preventing physical disorders in animals using these ligands.
2. Background Art
Protein Domain Analysis. Large proteins are typically made up of more than one domain and sometimes more than one subunit. This can complicate structure determination by X-ray crystallography if an attribute of any one part of the molecule is inhibitory to crystallization. Sometimes the overall flexibility of the multidomain structure can make it difficult for the protein to crystallize. In addition, large proteins (>40 kDa) are out of the range for structure determination by NMR techniques. One approach to circumvent these problems is to try to split the protein into domains and determine the structure in a piece-by-piece fashion. Here it is helpful to have information from sequence comparisons and or partial proteolysis to delineate the domain boundaries.
Calpains. The conventional calpains, better known as the m- and μ-calpains, are mammalian cellular cysteine proteases activated by Ca2+. They are the founding members of the calpain superfamily, which contains isoforms from mammals to various organisms such as Drosophila melanogaster and Caenorhabditis elegans (Sorimachi, H. and Suzuki, K. J. Biochem. (Tokyo) 129:653–664 (2001)). They function in Ca2+ signaling by modulating biological activities of their substrates through limited proteolysis (Sorimachi, H. and Suzuki, K. J. Biochem. (Tokyo) 129:653–664 (2001)). The conventional calpains are indispensable during development as indicated by knockout mice lethality (Arthur, J. S., et al., Mol. Cell Biol. 20:4474–4481 (2000); Zimmerman, U. J., et al., IUBMB. Life 50:63–68 (2001)) and they have been implicated in apoptosis (Wang, K. K., Trends Neurosci. 23:59 (2000)), cell cycle (Santella, L., et al. Cell Calcium 23:123–130 (1998)), and cell motility (Cox, E. A. and Huttenlocher, A., Microsc. Res. Tech. 43:412–419 (1998)). While physiological Ca2+ levels inside the cell are too low (<1 μM) for uncontrolled activation of either m- (>100 μM) or μ-calpain (>5 μM) (Croall, D. E., and DeMartino, G. N., Physiol Rev. 71:813–847 (1991)), during certain pathological states cellular Ca2+ levels can increase enough to achieve calpain activation without the aid of putative endogenous activators. Under such circumstances unrestrained proteolysis by calpains can result in tissue damage seen during ischemic injury (heart, brain)(Wang, K. K., and Yuen, P. W., Trends Pharmacol. Sci. 15: 412–419 (1994); Lee, K. S., et al., Ann. N. Y. Acad. Sci. 825: 95–103 (1997)) and neurodegeneration (Alzheimer's disease) (Patrick, G. N., et al. Nature 402: 615–622 (1999); Lee, M.S., et al. Nature 405: 360–364 (2000); Nixon, R. A., Ann. N. Y. Acad. Sci. 924: 117–131 (2000)). Administering existing calpain inhibitors has proven to lessen or prevent the onset of such conditions, but the lack of specific calpain inhibitors weakens the effectiveness of such therapies (Wang, K. K., and Yuen, P. W., Trends Pharmacol. Sci. 15: 412–419 (1994)).
The crystal structures of rat (Hosfield, C. M., et al., EMBO J 18:6880–6889 (1999)) and human (Strobl, S., et al., Proc. Natl. Acad. Sci. U.S.A. 97:588–592 (2000)) m-calpain heterodimers determined in the absence of Ca2+ have revealed a circular arrangement of domains. The circle extends from the anchor peptide (˜20 residues) at the N terminus of the large subunit (80 kDa), through the cysteine protease region (domains I˜190 residues and II˜145 residues), along the C2-like domain III (˜160 residues), down the linker (˜15 residues) and into the EF-hand-containing domain IV (˜170 residues). Domain W makes intimate contacts with the homologous 28 kDa small subunit (domain VI) through pairing of their fifth EF-hands, and the small subunit completes the ring by binding to the anchor peptide. Domain V of the small subunit is invisible in the human heterodimer structure likely due to its high content of glycine residues. In this circular structure, domains I and II are held slightly apart and miss-aligned such that the active site cleft is too wide for catalysis. Activation by Ca2+ must realign domains I and II to bring the catalytic residues in register for peptide bond hydrolysis. However, in the absence of a Ca2+-bound crystal structure the mechanism of activation of calpain remains controversial (Sorimachi, H. and Suzuki, K. J. Biochem. (Tokyo) 129: 653–664 (2001)). Although it is not clear if, and how, Ca2+ binding to the EF-hand domains initiates activation, some of the early events in this process, such as the autoproteolytic removal of the anchor peptide and/or the release of the small subunit, break the protein circle and lead to a general increase in susceptibility to proteolysis (Moldoveanu, T., et al., Biochim. Biophys. Acta 1545: 245–254 (2001)). It has heretofore been unknown whether these conformational changes release the constraints on domains I and II and allow them to form an active protease.
The difficulty in solving the structure of the calpain heterodimer in the presence of Ca2+ arises from subunit dissociation, often followed by large subunit aggregation under crystallization conditions. Nevertheless, a desirable template for rational drug design would be the assembled active site. The present invention provides such assembled active sites, and methods for producing and using such active sites.